The olive leaf extract oleuropein exerts protective effects against oxidant-induced cell death, concurrently displaying pro-oxidant activity in human hepatocarcinoma cells

Abstract

Objectives: Oleuropein (OP), the predominant natural constituent of leaves of the olive tree, exerts anti-inflammatory and antioxidant effects. The purpose of this study was to assess the protective effects of OP under the conditions of paraquat (PQ)-induced oxidative stress in vitro, using the human hepatocarcinoma cell line, HepG2.

Methods: Cell viability and death were determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and 4',6-diamidino-2-phenylindole-propidium iodide staining, respectively. Superoxide anion and lipid peroxidation levels were evaluated using nitroblue tetrazolium and thiobarbituric acid-reactive substances assays, respectively. Apoptosis was assessed by measuring poly(ADP-ribose) polymerase (PARP) and caspase-3 (Casp-3) cleavage via immunoblotting and immunofluorescence analyses.

Results: PQ induced a decrease in cellular viability by promoting necrosis through a mechanism involving superoxide generation and nuclear translocation of cleaved Casp-3. Co-treatment with OP afforded significant protection against the suppressive effects of PQ, as evident from increased cell viability, reduction of Casp-3 immunofluorescence, and normalization of β-tubulin expression levels. Unexpectedly, these OP-mediated protective effects were associated with increased superoxide and malondialdehyde generation and PARP cleavage.

Discussion: OP protects HepG2 cells against PQ-induced necrosis by suppressing Casp-3 cleavage while concomitantly acting as a pro-oxidant agent. This paradoxical mechanism of action of OP requires further investigation.

Keywords: Apoptosis; Hydroxyl radical; Lipid peroxidation; Necrosis; Oleuropein; Oxidative stress; Paraquat; Superoxide.

Figure 1

Measurement of HepG2 cellular viability with the MTT assay following 24 hours treatment with (A) increasing concentrations of oleuropein and (B) PQ with or without oleuropein. Results are expressed as means ± SEM of four to six independent experiments, with at least three replicates each. **P < 0.01 vs. PQ-treated cells.

Figure 2

OP potentiates oxidant production in HepG2 cells. (A) Measurement of superoxide anion production with the NBT assay in cells treated with PQ, OP, or both for 24 hours. (B) Measurement of lipid peroxidation (MDA) with the TBARS assay in cells treated with FeSO₄ + H₂O₂, OP, or both for 24 hours. Results are expressed as mean ± SEM of six independent experiments for NBT and three independent experiments for MDA. *P < 0.05 and **P < 0.01 vs. untreated control.

Figure 3

Effects of OP on PQ-induced changes in the levels of β-tubulin expression and PARP cleavage in HepG2 cells. (A) Western blot image. (B) Densitometric analysis of PARP expression. (C) Densitometric analysis of β-tubulin expression. Results were normalized using the corresponding GAPDH values. Data represent means ± SEM, n = 4. *P < 0.05 and **P < 0.01 vs. untreated control, ANOVA, Dunnett's post-test.

Figure 4

Cleaved caspase-3 immunofluorescence in HepG2 cells treated with OP (100 µmol/l), PQ (0.5 mmol/l) or both for 24 hours. (A) Confocal images and (B) analysis of corrected total cell fluorescence.